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Self-Assembly and Anti-Amyloid Cytotoxicity Activity of Amyloid Beta Peptide Derivatives V

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Self-Assembly and Anti-Amyloid Cytotoxicity Activity of Amyloid Beta Peptide Derivatives V Self-Assembly and Anti-Amyloid Cytotoxicity Activity of Amyloid beta Peptide Derivatives V. Castelletto, P. Ryumin, R. Cramer, I. W. Hamley, M. Taylor, D. Allsop, M. Reza, J. Ruokolainen, T. Arnold, D. Hermida-Merino, et al. To cite this version: V. Castelletto, P. Ryumin, R. Cramer, I. W. Hamley, M. Taylor, et al.. Self-Assembly and Anti- Amyloid Cytotoxicity Activity of Amyloid beta Peptide Derivatives. Scientific Reports, Nature Pub- lishing Group, 2017, 7, 12 p. 10.1038/srep43637. hal-01691906 HAL Id: hal-01691906 https://hal.archives-ouvertes.fr/hal-01691906 Submitted on 24 Jan 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. www.nature.com/scientificreports OPEN Self-Assembly and Anti-Amyloid Cytotoxicity Activity of Amyloid beta Peptide Derivatives Received: 30 September 2016 V. Castelletto1, P. Ryumin1, R. Cramer1, I. W. Hamley1, M. Taylor2, D. Allsop2, M. Reza3, Accepted: 26 January 2017 J. Ruokolainen3, T. Arnold4, D. Hermida-Merino5, C. I. Garcia6, M. C. Leal6 & E. Castaño6 Published: 08 March 2017 The self-assembly of two derivatives of KLVFF, a fragment Aβ(16–20) of the amyloid beta (Aβ) peptide, is investigated and recovery of viability of neuroblastoma cells exposed to Aβ (1–42) is observed at sub- stoichiometric peptide concentrations. Fluorescence assays show that NH2-KLVFF-CONH2 undergoes hydrophobic collapse and amyloid formation at the same critical aggregation concentration (cac). In contrast, NH2-K(Boc)LVFF-CONH2 undergoes hydrophobic collapse at a low concentration, followed by amyloid formation at a higher cac. These findings are supported by theβ -sheet features observed by FTIR. Electrospray ionization mass spectrometry indicates that NH2-K(Boc)LVFF-CONH2 forms a significant population of oligomeric species above thecac . Cryo-TEM, used together with SAXS to determine fibril dimensions, shows that the length and degree of twisting of peptide fibrils seem to be influenced by the net peptide charge. Grazing incidence X-ray scattering from thin peptide films shows features of β-sheet ordering for both peptides, along with evidence for lamellar ordering of NH2-KLVFF- CONH2. This work provides a comprehensive picture of the aggregation properties of these two KLVFF derivatives and shows their utility, in unaggregated form, in restoring the viability of neuroblastoma cells against Aβ-induced toxicity. The Amyloid β (Aβ ) peptide plays a key role in Alzheimer’s disease (AD), which is an increasingly prevalent con- dition in the aging population and as such is a major global healthcare challenge. There is thus intense research activity on the role of the Aβ peptide in causing neuronal cell death and in the progression of AD and its eventual treatment1–10. It is believed that aggregates of Aβ peptide, misfolded into β -sheet fibrils (so-called “amyloid”) are implicated in this process since clumps of aggregates termed plaques have been observed in brain tissue from AD sufferers. Oligomeric aggregates are believed to have higher cytotoxicity than fibrillar aggregates11–15. One strategy to potentially treat AD is to hinder or disrupt aggregation, or more specifically to disrupt the formation of oligomers. A key sequence driving aggregation in Aβ is the core sequence Aβ 16–20, KLVFF, which contains the diphe- nylalanine sequence which plays a significant effect in its aggregation propensity10. The aggregation of KLVFF has been investigated previously16–28 and many variants have been prepared in order to examine their influence on the aggregation of Aβ itself10,29. In a previous paper we examined the self-assembly of the KLVFF peptide 30 with uncapped termini, NH2-KLVFF-COOH , and showed for the first time clear evidence (in particular using cryogenic-TEM and small-angle X-ray scattering) that this molecule itself forms highly extended fibrils, with β-sheet structure (confirmed via FTIR spectroscopy and X-ray diffraction). The CD spectrum is dominated by the contribution of the π-stacking phenylalanine units which masks the typical spectrum of a β-sheet structure10,29,30. Some of us have previously reported a peptide inhibitor, (Ac-rGFFVLKGr-NH2, r denotes d-arginine) consisting of the retro-inverted version of KLVFF flanked by the solubilizing residues rG and Gr, that blocks the formation of Aβ oligomers and fibrils in vitro and also inhibits the toxic effects of Aβ on cell cultures31. 1School of Chemistry, Pharmacy and Food Biosciences, University of Reading, Whiteknights, Reading RG6 6AD, UK. 2Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster LA1 4YQ, UK. 3Department of Applied Physics, Aalto University School of Science, Aalto FI-00076, Finland. 4Diamond Light Source Ltd., Harwell Science and Innovation Campus, Didcot OX11 0DE, UK. 5European Synchrotron Radiation Facility, ESRF, 71 avenue des Martyrs, 38000 Grenoble, France. 6Fundación Instituto Leloir and Instituto de Investigaciones Bioquímicas de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina. Correspondence and requests for materials should be addressed to I.W.H. (email: I.W.Hamley@ reading.ac.uk) SCIENTIFIC REPORTS | 7:43637 | DOI: 10.1038/srep43637 1 www.nature.com/scientificreports/ Figure 1. Molecular structures of peptide 1 and peptide 2. Here, we examine the self-assembly of the KLVFF peptide with C-terminal amidation, NH2-KLVFF-CONH2 (peptide 1) compared to the lysine capped analogue NH2-K(Boc)LVFF-CONH2 (peptide 2). The two peptides are shown in Fig. 1. In the latter peptide the lysine residue is capped with a Boc (tert-butyloxylcarbonyl) unit. In order to examine the influence of the lysine group and electrostatics on aggregation behaviour and bioactivity, we compare the self-assembly and cytotoxicity of peptide 1 with that of the homologue peptide 2. A range of spectroscopy, microscopy and scattering techniques are used to investigate peptide self-assembly. Electrospray ionisation mass spectrometry is used to probe oligomeric species. Neurotoxicity assays are performed on Aβ (1–42) /peptide mixtures in order to assess the utility of these samples as therapeutic agents. Results Critical Aggregation Concentration. Two types of fluorescence assays were used to determine critical aggregation concentration (cac) values. The first uses ANS, the fluorescence of which depends on the hydrophobic environment32–36. The second uses ThT, which is sensitive to the formation of amyloid fibrils32,37–40. Results of the two fluorescence assays for peptide 1 are shown in Fig. 2. The cac value obtained from the discontinuity in fluores- cence intensity using ThT is in agreement within uncertainty with that from the ANS assay (1.00 ± 5 × 10−2) wt%. This indicates that for this peptide, hydrophobic collapse occurs concurrently with the formation of amyloid fibrils. The concentration-dependent fluorescence results for peptide 2 are shown in Fig. 3. The data shows firstly that the cac is substantially lower than for peptide 1, due to the presence of the additional hydrophobic Boc group. In addition, significantly different values are obtained for the cac from the two fluorescence assays. This suggests that hydrophobic collapse occurs before formation of amyloid fibrils for sample 2 and that in between the two cac values (5.9 ± 0.1) × 10−3 wt% and (1.9 ± 0.1) × 10−2 wt% oligomers may be present. Electrospray Ionization Mass Spectrometry. ESI experiments were performed at 0.029 wt% to investi- gate the peptide’s aggregation stability. According to the results above, 0.029 wt% is below the cac for 1 but at the onset of peptide aggregation for 2. ESI studies were first performed for peptides dissolved in water. Subsequently, the same study was repeated for peptides dissolved in HFIP: water (1:9; v/v). HFIP was added to test the stability of the peptide aggregates, since this organic solvent is routinely used to disrupt Aβ oligomerization in solution41. ESI mass spectra showing peptide aggregation stability for 1 and 2 with and without HFIP are displayed in Fig. 4. Figures S1 and S2 show the ESI mass spectra with further peak annotation for peptide aggregation for 1 and 2, respectively. Figures 4a,c and S1 show that 1 at a concentration of 0.029 wt% forms only a few self-assemblies of low aggre- gation state (up to n =​ 7). The relative extents of oligomerisation of 1 measured in experiments without and with HFIP are (Eo)H2O = 0.040 ±​ 0.002 and (Eo)HFIP = 0.046 ±​ 0.002, respectively. This data shows that the oligomers at such low concentrations are stable upon HFIP addition. Figures 4b,d show that at 0.029 wt% 2 forms more SCIENTIFIC REPORTS | 7:43637 | DOI: 10.1038/srep43637 2 www.nature.com/scientificreports/ Figure 2. Critical aggregation concentration (cac) assay for peptide 1 using concentration-dependent (a) ANS and (b) ThT fluorescence assays. Figure 3. Critical aggregation concentration (cac) assay for peptide 2 using concentration-dependent (a) ANS and (b) ThT fluorescence assays. oligomers than 1 at a much higher aggregation state (up to n =​ 20) which are not stable upon HFIP addition. The corresponding relative extents of oligomerisation of 2 measured in experiments without and with HFIP are (Eo)H2O = 0.25 ±​ 0.002 and (Eo)HFIP = 0.027 ±​ 0.004, respectively. As a whole, the ESI results are consistent with the cac assays in Figs 2 and 3. They show that there is little peptide aggregation for 1 below the cac, while for 2 extensive oligomerisation starts at this concentration.
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